JC virus-induced Progressive Multifocal Leukoencephalopathy

Progressive multifocal encephalopathy (PML) is a fatal demyelinating disease of the central nervous system (CNS), caused by the lytic infection of oligodendrocytes by a human polyomavirus, JC virus (JCV). PML is rare disease but mostly develops in patients with underlying immunosuppressive conditions, including Hodgkin's lymphoma, lymphoproliferative diseases, in those undergoing antineoplastic therapy and AIDS. However, consistent with the occurrence of PML under immunocompromised conditions, this disease seems to be also steadily increasing among autoimmune disease patients (multiple sclerosis and Crohn's disease), who are treated with antibody-based regimens (natalizumab, efalizumab and rituximab). This unexpected occurrence of the disease among such a patient population reconfirms the existence of a strong link between the underlying immunosuppressive conditions and development of PML. These recent observations have generated a new interest among investigators to further examine the unique biology of JCV.     

Meta-omics analysis indicates the saliva microbiome and its proteins associated with the prognosis of oral cancer patients

Saliva is a biofluid that maintains the health of oral tissues and the homeostasis of oral microbiota. Studies have demonstrated that Oral squamous cell carcinoma (OSCC) patients have different salivary microbiota than healthy individuals. However, the relationship between these microbial differences and clinicopathological outcomes is still far from conclusive. Herein, we investigate the capability of using metagenomic and metaproteomic saliva profiles to distinguish between Control (C), OSCC without active lesion (L0), and OSCC with active lesion (L1) patients. The results show that there are significantly distinct taxonomies and functional changes in L1 patients compared to C and L0 patients, suggesting compositional modulation of the oral microbiome, as the relative abundances of Centipeda, Veillonella, and Gemella suggested by metagenomics are correlated with tumor size, clinical stage, and active lesion. Metagenomics results also demonstrated that poor overall patient survival is associated with a higher relative abundance of Stenophotromonas, Staphylococcus, Centipeda, Selenomonas, Alloscordovia, and Acitenobacter. Finally, compositional and functional differences in the saliva content by metaproteomics analysis can distinguish healthy individuals from OSCC patients. In summary, our study suggests that oral microbiota and their protein abundance have potential diagnosis and prognosis value for oral cancer patients. Further studies are necessary to understand the role of uniquely detected metaproteins in the microbiota of healthy and OSCC patients as well as the crosstalk between saliva host proteins and the oral microbiome present in OSCC.     

Crystal structure of human PACRG in complex with MEIG1 reveals roles in axoneme formation and tubulin binding

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Loss of CDK5RAP2 affects neural but not non-neural mESC differentiation into cardiomyocytes

Biallelic mutations in the gene encoding centrosomal CDK5RAP2 lead to autosomal recessive primary microcephaly (MCPH), a disorder characterized by pronounced reduction in volume of otherwise architectonical normal brains and intellectual deficit. The current model for the microcephaly phenotype in MCPH invokes a premature shift from symmetric to asymmetric neural progenitor-cell divisions with a subsequent depletion of the progenitor pool. The isolated neural phenotype, despite the ubiquitous expression of CDK5RAP2, and reports of progressive microcephaly in individual MCPH cases prompted us to investigate neural and non-neural differentiation of Cdk5rap2-depleted and control murine embryonic stem cells (mESC). We demonstrate an accumulating proliferation defect of neurally differentiating Cdk5rap2-depleted mESC and cell death of proliferative and early postmitotic cells. A similar effect does not occur in non-neural differentiation into beating cardiomyocytes, which is in line with the lack of non-central nervous system features in MCPH patients. Our data suggest that MCPH is not only caused by premature differentiation of progenitors, but also by reduced propagation and survival of neural progenitors.     

Production of N-sulfated polysaccharides using yeast-expressed N-deacetylase/N-sulfotransferase-1 (NDST-1)

Heparan sulfate/heparin N-deacetylase/N-sulfotransferase-1 (NDST-1) is a critical enzyme involved in heparan sulfate/heparin biosynthesis. This dual-function enzyme modifies the GlcNAc-GlcA disaccharide repeating sugar backbone to make N-sulfated heparosan. N-sulfation is an absolute requirement for the subsequent epimerization and O-sulfation steps in heparan sulfate/heparin biosynthesis. We have expressed rat liver (r) NDST-1 in Saccharomyces cerevisiae as a soluble protein. The yeast-expressed enzyme has both N-deacetylase and N-sulfotransferase activities. N-acetyl heparosan, isolated from Escherichia coli K5 polysaccharide, de-N-sulfated heparin (DNSH) and completely desulfated N-acetylated heparan sulfate (CDSNAcHS) are all good substrates for the rNDST-1. However, N-desulfated, N-acetylated heparin (NDSNAcH) is a poor substrate. The rNDST-1 was partially purified on heparin Sepharose CL-6B. Purified rNDST-1 requires Mn(2+) for its enzymatic activity, can utilize PAPS regenerated in vitro by the PAPS cycle (PAP plus para-nitrophenylsulfate in the presence of arylsulfotransferase IV), and with the addition of exogenous PAPS is capable of producing 60-65% N-sulfated heparosan from E. coli K5 polysaccharide or Pasteurella multocida polysaccharide.     

ZmMYB31 directly represses maize lignin genes and redirects the phenylpropanoid metabolic flux

Few regulators of phenylpropanoids have been identified in monocots having potential as biofuel crops. Here we demonstrate the role of the maize (Zea mays) R2R3-MYB factor ZmMYB31 in the control of the phenylpropanoid pathway. We determined its in vitro consensus DNA-binding sequence as ACC(T)/(A) ACC, and chromatin immunoprecipitation (ChIP) established that it interacts with two lignin gene promoters in vivo. To explore the potential of ZmMYB31 as a regulator of phenylpropanoids in other plants, its role in the regulation of the phenylpropanoid pathway was further investigated in Arabidopsis thaliana. ZmMYB31 downregulates several genes involved in the synthesis of monolignols and transgenic plants are dwarf and show a significantly reduced lignin content with unaltered polymer composition. We demonstrate that these changes increase cell wall degradability of the transgenic plants. In addition, ZmMYB31 represses the synthesis of sinapoylmalate, resulting in plants that are more sensitive to UV irradiation, and induces several stress-related proteins. Our results suggest that, as an indirect effect of repression of lignin biosynthesis, transgenic plants redirect carbon flux towards the biosynthesis of anthocyanins. Thus, ZmMYB31 can be considered a good candidate for the manipulation of lignin biosynthesis in biotechnological applications.     

Differential Postnatal Development of Monoamine Oxidases A and B in the Blood-Brain Barrier of the Rat

Abstract: We studied the monoamine metabolizing mitochondrial enzyme, monoamine oxidase (MAO), in cerebral microvessels obtained from postnatally developing rats by measuring the specific binding of [³H]pargyline, an irreversible inhibitor of MAO, and the rate of oxidation of three known MAO substrates: benzylamine, 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine, and tryptamine. MAO activity increased postnatally, with the greatest increase occurring in the second week and reaching a peak at 3 weeks of age. A concomitant increase in MAO of the cerebral cortex also occurred, but was several-fold less than that of cerebral microvessels. Using clorgyline and deprenyl, relatively specific inhibitors of MAO-A and MAO-B, we showed that cerebral microvessels contain both forms of MAO at all ages, but there was a major preponderance in the postnatal development of MAO-B. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) analyses of rat microvessels after [³H]pargyline binding also showed two distinct bands of radioactivity at all ages. These two bands corresponded to molecular weights of ∼6.5,000 for MAO-A and -60,000 for MAO-B. SDS-PAGE resuits of brain microvessels obtained from 1-, 14-, and 42-day-old rats confirm the differential postnatal development of MAO-B in rat brain microvessels.     

TMBIM5 is the Ca2+/H+ antiporter of mammalian mitochondria

Mitochondrial Ca²⁺ ions are crucial regulators of bioenergetics and cell death pathways. Mitochondrial Ca²⁺ content and cytosolic Ca²⁺ homeostasis strictly depend on Ca²⁺ transporters. In recent decades, the major players responsible for mitochondrial Ca²⁺ uptake and release have been identified, except the mitochondrial Ca²⁺/H⁺ exchanger (CHE). Originally identified as the mitochondrial K⁺/H⁺ exchanger, LETM1 was also considered as a candidate for the mitochondrial CHE. Defining the mitochondrial interactome of LETM1, we identify TMBIM5/MICS1, the only mitochondrial member of the TMBIM family, and validate the physical interaction of TMBIM5 and LETM1. Cell‐based and cell‐free biochemical assays demonstrate the absence or greatly reduced Na⁺‐independent mitochondrial Ca²⁺ release in TMBIM5 knockout or pH‐sensing site mutants, respectively, and pH‐dependent Ca²⁺ transport by recombinant TMBIM5. Taken together, we demonstrate that TMBIM5, but not LETM1, is the long‐sought mitochondrial CHE, involved in setting and regulating the mitochondrial proton gradient. This finding provides the final piece of the puzzle of mitochondrial Ca²⁺ transporters and opens the door to exploring its importance in health and disease, and to developing drugs modulating Ca²⁺ exchange.     

Anaerobic membrane reactor with phase separation for the treatment of cheese whey

Two-phase anaerobic digestion of cheese whey was investigated in a system consisting of a stirred acidogenic reactor followed by a stirred methanogenic reactor, the latter being coupled to a membrane filtration system to enable removal of soluble effluent whilst retaining solids. The acidogenic reactor was operated at a hydraulic retention time (HRT) of one day, giving maximum acidification of 52.25% with up to 5 g/l volatile fatty acids, of which 63.7% was acetic acid and 24.7% was propionic acid. The methanogenic reactor received an organic load up to 19.78 g COD/ld, corresponding to a HRT of 4 days, at which 79% CODs and 83% BOD(5) removal efficiencies were obtained. Average removals of COD, BOD(5) and TSS in the two-phase anaerobic digestion process were 98.5%, 99% and 100%, respectively. The daily biogas production exceeded 10 times reactor volume and biogas methane content was greater than 70%.     

Refolding of human beta-1-2 GlcNAc transferase (GnT1) and the role of its unpaired Cys 121

Human beta1-2N-acetylglucosaminyltransferase (hGnT1) lacking the first 103 amino acids was expressed as a maltose binding protein (MBP) fusion protein in inclusion bodies (IBs) in Escherichia coli and refolded using an oxido-shuffling method. GnT1 mutants were prepared by replacing a predicted unpaired cysteine (C121) with alanine (C121A), serine (C121S), threonine (C121T) or aspartic acid (C121D). A double mutant R120A/C121H, was generated to mimic Gly14, the Caenorhabditis elegans GnT1 counterpart to hGNT1. Each mutant hGnT1 was constructed as an MBP fusion protein and resultant IBs were isolated and refolded. Wild type hGnT1 and mutants C121A, C121S and R120A/C121H transferred UDP-GlcNAc to the glycoprotein acceptor Man(5)-RNAse B, whereas mutants C121T and C121D were inactive. These findings indicated that cysteine 121 has a structural role in maintaining active site geometry of hGnT1, rather than a catalytic role, and illustrates for the first time the potential utility of E. coli as an expression system for hGnT1.